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provided by Wageningen University & Research Publications BioControl DOI 10.1007/s10526-008-9189-2

Hyperparasitism behaviour of the autoparasitoid tricolor on two secondary host species

Ying Huang Æ Antoon J. M. Loomans Æ Joop C. van Lenteren Æ Xu RuMei

Received: 19 November 2006 / Accepted: 23 July 2008 Ó International Organization for Biological Control (IOBC) 2008

Abstract Hyperparasitism by virgin female Encar- However, rates of hyperparastism were different sia tricolor was studied by direct observation of its according to host stage and host species. Hosts in behaviour when contacting two secondary host the late larval stages were most preferred for species ( and E. tricolor) at diffe- hyperparasitization and the heterospecific E. formosa rent host stages (first and second larval stage, third was more preferred as a secondary host than the larval stage, and pupal stage). The searching and conspecific, E. tricolor, in particular from the prepu- hyperparasitism behavioural sequence of E. tricolor pal stage onwards. The window of vulnerability, i.e., was independent of the host stage of the whitefly the duration of the period in which a secondary host (Aleyrodes proletella), and was similar to several is susceptible to hyperparasitism, was largely deter- related primary parasitoid species. In experiments mined by the occurrence and rate of melanization with equal numbers of secondary hosts, encounter after the onset of pupation. The duration of a frequencies were equal for both secondary host successful hyperparasitization event was longer than species in all developmental stages observed. one that failed. Superparasitism occurred only once in all cases. The potential effect of autoparasitoids on biological control programs and the consequences for Handling editor: Torsten Meiners. selection and release of an effective, yet ecologically safe agent are discussed. Y. Huang Institute of and Plant Quarantine, Chinese Academy of Inspection and Quarantine, Beijing, Peoples Keywords Encarsia formosa Á Hymenoptera Á Republic of China Aphelinidae Á Autoparasitoid Á Hyperparasitoid Á Behaviour Á Aleyrodes proletella Á Y. Huang Á A. J. M. Loomans (&) Á J. C. van Lenteren Laboratory of Entomology, Wageningen University, Environmental effects Wageningen, The Netherlands e-mail: [email protected]

Y. Huang Á X. RuMei College of Life Science, Beijing Normal University, Introduction Beijing, Peoples Republic of China It is commonly believed that the application of Present Address: biological control is a safe alternative to pesticides, A. J. M. Loomans Department of Entomology, Plant Protection Service, and a wide range of parasitoids has been released Wageningen, The Netherlands successfully as biological control agents (Gurr and 123 Y. Huang et al.

Wratten 2000). Several species of aphelinid parasi- parasitoids (hyperparasitoids) on larvae or pupae of toids have been used to help suppress populations their own or other primary parasitoid species. Mated of the two most economically important whitefly female autoparasitoids may lay both fertilized and species, Trialeurodes vaporariorum (Westwood) unfertilized eggs, but virgin females can only lay (greenhouse whitefly) and Bemisia tabaci (Gennadius) unfertilized eggs in secondary hosts (Gerling 1966). (tobacco whitefly) (both : Aleyrodidae; It is generally thought that parasitoids with hyper- e.g. Gerling et al. 2001; van Lenteren and Woets parasitic behaviour are injurious in biological control 1988; van Lenteren et al. 1996). A number of these programs (Luck et al. 1981) and it is standard parasitoids are members of the genus Encarsia quarantine procedure to exclude exotic obligate (Fo¨rster), such as Encarsia formosa (Gahan) hyperparasitoids from biological control programmes (Hymenoptera: Aphelinidae). Whereas the economic (Sullivan and Vo¨lkl 1999). However, several auto- benefits are clear, the ecological effects of an introduced parasitoid species have been successfully introduced species on the indigenous fauna are not. During the past as biological control agents (Bogra´n and Heinz decades, Howarth (1991) and others argued that the 2002), some introductions have, however, resulted import and release of exotic species for biological in problems, such as those of Encarsia pergandiella control might create problems for the indigenous fauna. Howard. The latter species was imported into Italy to Recent reviews show that such exotic natural enemies control the greenhouse whitefly, but established have in some cases causednegative effects on non-target outside, and can now be found all around the organisms and the environments (Louda et al. 2003; Mediterranean Area (Portugal, Spain, Italy, France, van Lenteren et al. 2006). Tunesia) (Loomans and van Lenteren 1999), With regard to biological control of exotic green- regionally upsetting successful biological control house whiteflies in Europe, the introduced species applications by primary parasitoids in greenhouses E. formosa may encounter native species of whiteflies (Gabarra et al. 1999, 2003). In New Zealand there as well as native parasitoids, like Encarsia tricolor have been several instances where E. pergandiella (Fo¨rster). The possibility of such interactions raises has been present in significant numbers on green- several questions: Will the exotic and indigenous house tomato crops and although large introductions parasitoids coexist, or will one of them lose the of E. formosa were made weekly, control of the pest competition and will it be displaced? What will the was lost (John Thompson, personal communication effect be on the dynamics of indigenous whitefly 2005). populations? And what kind of effect has the exotic The host range of autoparasitoids is much wider biological control agent on the indigenous ecosystem? and multitrophic effects are larger than that of These questions are related to the interactions between primary parasitoids, because of their heteronomous species of parasitoids at the level of parasitoid hyperparasitoid behaviour. Therefore, the concern behaviour, life history and the interaction between about the direct and indirect ecological effects of these parasitoids and their hosts (Murdoch 1996). autoparasitoids in biological control remains wide Another question we are faced with is what the open (Rosen 1981). Since autoparasitoids occupy two effect could be of the release of an exotic primary trophic levels, it is not proper to separate the overall parasitoid on the population dynamics and survival of interactions into several two-species interactions facultative autoparasitoids. This question relates to (host-parasitoid or primary-hyperparasitoid) (Hassell the rather typical biology of several aphelinid para- 2000). We propose to study such relationships from sitoids (e.g. Hunter and Kelly 1998). Males and two viewpoints: (1) the effect of two competing females of hymenopteran parasitoid species in the parasitoids for one primary host; (2) and the effects of Aphelinidae develop in or on different kinds of hosts, the interactions between a primary and a hyper- and are therefore called heteronomous hyperparasi- parasitoid (secondary parasitoid) (Fig. 1; May and toids, more specifically autoparasitoids (Hunter and Hassell 1981). Woolley 2001). Fertilized female eggs develop as In the current study we considered the relationship obligate primary parasitoids on their primary hosts, between an exotic biological control agent (the whiteflies or scale . On the other hand, strictly primary parasitoid E. formosa) and a native unfertilized male eggs develop as secondary parasitoid (the facultative autoparasitoid E. tricolor) 123 Hyperparasitism behaviour

comparison of the behavioural strategy towards conspecific and heterospecific secondary hosts may facultative be used also to clarify the issues on the role of autoparasitoid autoparasitoids in biological control mentioned above. From both systems mentioned (Avilla and Copland 1987; Williams 1991)—directly or, as in the

unmated Avilla’s paper, indirectly—a preference emerged of E. tricolor females towards heterospecific secondary hosts for male egg oviposition. Behavioural observa- tions will assist in testing the hypothesis that primary parasitoid autoparasitoids can discriminate between species of (secondary host) secondary hosts to reduce self-hyperparasitism and henceforth this preference may negatively affect the outcome of a biological control programme. We specifically studied the host preference of the native autoparasitoid E. tricolor when offered different secondary host species, and the effect that different life stages of the host may have on this preference. primary host

Materials and methods

Fig. 1 Diagram to illustrate the relationships of a three- and plant rearing species system, which is containing an autoparasitoid (after May and Hassell 1981) The cabbage whitefly, Aleyrodes proletella, was used as primary host. It was cultured on cabbage (Brussels on a native host (the cabbage whitefly, Aleyrodes sprouts, oleracea gemmifera cv. Cyrus) in a proletella L.). The autoparasitoid E. tricolor is widely greenhouse at 21°C and 16L:8D. As primary parasi- distributed throughout the West Palaearctic region. toids we used E. formosa and E. tricolor females. Females are solitary endoparasitoids of several E. formosa was obtained from a commercial company whitefly species, including A. proletella, Aleurotuba (EnStripÒ, Koppert Biological Systems, The Nether- jelinekii (Frauenfeld) and T. vaporariorum (Williams lands) and was reared on cabbage with A. proletella 1995). Males are solitary endoparasitoids of primary in ventilated plastic cages (45 9 30 9 35 cm). parasitoids of whiteflies, such as E. inaron (Walker), E. tricolor was collected from cabbage fields E. formosa and females of its own kind (Avilla and (Wageningen, The Netherlands) and also reared in Copland 1987; Williams 1991, 1996). Both the cages on cabbage and cabbage whitefly. During the primary female and secondary male have the following rearing process, clip cages (20 mm diameter) were developmental stages: egg, first, second and third used to introduce a certain number of whiteflies or instar larva, prepupa and pupa. The developmental parasitoids to the target leaves. rate of both the male and female immature stages of the parasitoid is influenced by the host stages which Host choice tests are parasitized (Williams 1995) and the male has a shorter developmental time than the female (Avilla Whiteflies were confined to the underside of leaves and Copland 1987). Until now, no direct behavioural inside clip cages for 24 h, thus allowing the new records of hyperparasitic behaviour in E. tricolor generation of whitefly individuals to develop almost have been reported, though these kind of observations synchronously. After 14–15 days, when immature assist in evaluating the efficiency of natural enemies whiteflies were in the late third (L3) to early fourth in biological control programmes (van Roermund (L4) nymphal stage, primary parasitoids, E. formosa et al. 1996). Direct observations and subsequent or E. tricolor, were introduced in clip cages, and 123 Y. Huang et al. plants were moved into a climate cell at 25°C. host, turning on a host, drilling a host with the Introduced wasps were removed after 24 h. After ovipositor, host feeding, honeydew feeding, walking, some days, when secondary larval parasitoid hosts standing (including standing still and standing with were present, most unparasitized whiteflies had preening) and jumping (van Lenteren et al. 1980). An already emerged, or were in the red-eyed stage and observation was aborted when: (1) the wasp spent could be easily recognized and removed. It is difficult more than 15 min standing still on the leaflet or left to recognize parasitoid larvae as such from the the leaflet immediately after introduction, or (2) the outside through the whitefly cuticle when they are wasp encountered all the provided hosts within the still in their early instar stages. By removing unpa- maximum period of 2 h. After the observation period, rasitized whiteflies we avoided providing primary all hosts were dissected to examine whether parasi- whitefly hosts instead of secondary parasitoid hosts to toid eggs were present. Parasitoids that left the E. tricolor during the experiments. Parasitoid imma- leaf-surface during the observation period without tures (secondary hosts) in the late larval or prepupal displaying any searching behaviour, were not considered stage can easily be recognized by their shape through valid records and were excluded from data analysis. the transparent whitefly cuticle (primary hosts). The justification of our approach was also proven during Data analysis and statistical test dissection, where secondary host larvae were always found. Statistical tests were performed using SPSS 10 Choice tests were designed with the two secondary (ÓSPSS Inc. 1989–1999). Results of observations host species offered simultaneously in one arena. The involving different host stages were analyzed using hosts were parasitized whitefly nymphs containing an ANOVA test. A v2 test was used to compare the female E. tricolor and female E. formosa immatures differences between values for the two host types in equal numbers. Twelve hosts of each species in the within the same host stage. same stage were provided in one arena to an individual female of E. tricolor. A small drop of honey was applied as food source. Results Three treatments were performed including three different host stages: first and second instar larvae Description of searching and parasitization (L1 and L2), third instar larvae (L3), and prepupa. behaviour of E. tricolor Between 4 and 12 days after primary parasitoids were introduced, whitefly hosts containing secondary host The behaviour of virgin E. tricolor females can be larvae or prepupae inside were selected and moved to divided in two parts, searching on the leaf and a clean leaflet with a needle and glued with starch. handling of hosts. When virgin E. tricolor females This was done after 4–6 days to obtain L1 and L2 were introduced onto a leaflet, most of them began to secondary hosts, 8–9 days to obtain L3, and 10–12 search (walking and drumming), using their antennae. days to obtain prepupal hosts. The leaflet was placed They sometimes made short stops during which they on a piece of moist filter paper in a small Petri dish. either stood still, preened, or fed on the honey. The The size of the leaflets was about 2.5 9 2.0 cm, and following sequence of behavioural elements was the distance between the hosts was about 0.5 cm. exhibited when a host was found: encountering a Each treatment was repeated 8 or 9 times. host, drumming a host with antennae, turning on the For each treatment, the searching and host- host, drilling the host with the ovipositor (oviposition handling behaviour of 48–72 h old E. tricolor virgin posture) or leaving the host. Sometimes host-feeding females was observed under a stereoscope for a occurred after drilling a host. maximum period of 2 h. The whole process was No significant differences (P [ 0.1) were found in recorded by using a handheld computer and The any of the durations of the same behavioural element Observer (Noldus IT, Wageningen, The Netherlands) performed by adult females of E. tricolor on the two for registration and calculation of behavioural secondary hosts, E. formosa or E. tricolor (Fig. 2A). elements. The following behavioural elements we Therefore, the further descriptions of the general were distinguished: encountering a host, drumming a behaviour, such as time budget and behavioural 123 Hyperparasitism behaviour sequences, were made by combining the observations a short time (15.0 ± 1.4 s in E. formosa, 12.9 ± for the two secondary hosts. 0.8 s in E. tricolor). The duration in oviposition Durations of behavioural elements (seconds ± posture resulting in host rejection was much longer s.e.) dealing with a host were calculated from the (332.9 ± 44.8 s for E. formosa, 281.6 ± 36.4 s for moment of encountering a host till leaving that host E. tricolor). The time to accept a host and laying an (Fig. 2A) and include antennal rejection of the hosts egg was still longer (580.8 ± 73.2 s for E. formosa, (i.e. parasitoids left hosts after antennal testing 563.1 ± 120.1 s for E. tricolor). When host-feeding (drumming or drumming and turning)); ovipositor occurred, it always lasted longer than 25 min rejection (i.e. parasitoids inserted ovipositors into (1516.9 ± 142.1 s for E. formosa and 1770.6 s for hosts after antennal testing, but did not lay any egg); E. tricolor), and was quite different from the duration host acceptance (i.e. hyperparasitization, parasitoids of feeding on the honey, which took only about 2 min inserted their ovipositors into hosts, and laid a male (Fig. 2B). egg inside it); and host-feeding after oviposition Visits to the leaflet were generally short and consisted posture. Hosts that were rejected after antennal largely of walking, standing and honey feeding. The drumming were examined by the parasitoid only for average duration of walking was 23.0 ± 51.2 s,

Fig. 2 Average duration (time in seconds ± s.e.) of behavioural elements exhibited by virgin female E. tricolor when searching on leaflets and when handling hosts. The figures in brackets under the bars represent the number of times a certain behavioural element was recorded. (A) average duration (time in seconds ± s.e.) of behavioural elements (antennal rejection, ovipositor rejection, parasitization and host feeding) on different secondary hosts. (B) average duration (time in seconds ± s.e.) of behavioural elements (walking, standing and honey feeding) on leaflets

123 Y. Huang et al. duration of standing 28.9 ± 36.0 s, and duration of was the most frequently observed activity (1207 times), feeding on honey lasts 132.1 ± 16.1 s. the next in line was standing (605 times) and encoun- The time budget of E. tricolor in this set-up was as tering host (596 times). Drumming always followed follows: a wasp spent on average 66.9% of its time upon host encounter. More than half (56.4%) of host handling hosts, and 33.1% on searching, honey encounters and drumming was followed by turning on feeding and standing still (Fig. 3). More than half the host, and 60.7% of this turning was followed by (55.0%) of the total time of handling a host was spent insertion of the ovipositor. After oviposition, females on oviposition behaviour. often left the hosts (70.1%) and started walking. A simplified diagram showing the behavioural However, in 26.0% of the cases they stayed on the host sequences observed is presented in Fig. 4. Walking preening for a while before leaving.

Fig. 3 Time budget of E. tricolor when exposed to hosts on part of a leaf in a Petri dish. Total time left, host handling on the right

Fig. 4 Diagram of behavioural sequences of virgin E. tricolor D = drumming; T = turning on host; O = oviposition pos- females as observed in a Petri dish when exposed to equal ture; J = jumping; X = start/terminate/out of arena. Numbers number of heterospecific and conspecific secondary hosts show the percentages of behavioural elements involved, (n = 25): W = walking; S = standing (still or preening); between brackets are the absolute numbers of behavioural F = feeding (on host or honey); E = host encounter; elements observed 123 Hyperparasitism behaviour

In a few cases (3.9%), hosts were used for host- (Fig. 5). This means that the heterospecific secondary feeding after finishing the oviposition posture: wasps hosts are more preferred than the conspecific ones. retracted their ovipositor, turned on the host to locate The average absolute number of eggs laid by an the hole they had made, and started feeding with their individual E. tricolor female was strongly dependent heads bent down. We observed that the sequence of on the host stage offered. When exposed to an array of drilling, turning and feeding on the host was repeated 24 early larval stages during 2 hours 1.0 ± 0.3 eggs often, probably to widen the hole or to make a new were found, while in third larval stages 6.3 ± 0.8 and one. in prepupa 2.7 ± 0.3 eggs were found. Superparasitism occurred only once in all experiments. Effect of the host stage and host species on hyperparasitism behaviour (2) Time budgets on hosts of different stages and species (1) Parasitism percentages in different host stages and host species Table 2 shows the time budget of E. tricolor females when dealing with hosts of different stages. The total The percentage of hosts encountered, ovipositor observation time of wasps on leaflets with the drilling (excluding host-feeding), male egg depo- different host species and stages was the same, and sition and host feeding on each type of host are the time budgets mentioned in Table 2 are all based shown in Fig. 5 and Table 1. All these percentages on this total time. A clear difference in relative time were calculated based on the number of hosts distributed amongst the different behavioural ele- provided as 100%. ments can be seen when referring to different host Parasitoids encountered secondary hosts in similar stages. On all hosts, most time was spent on drilling. numbers. Thus the percentages of hosts encountered The longest drilling times were found on the L3 stage were the same for the two host species and all stages, (E. formosa 47.0 ± 3.1%, E. tricolor 29.4 ± 2.7%) and varied between 72.2 ± 5.6% and 85.9 ± 8.0% compared to the other two stages. The fact that the (Table 1). Host stage clearly affected the parasitoids’ longest drilling times were found on the L3 stage was acceptance behaviour. When the encountered consistent with the highest number of hosts drilled secondary hosts were E. formosa in the L3 and and highest number of oviposition postures observed prepupal stages, the host drilling percentages on this stage (Table 1). averaged 83.3 and 77.1% respectively, and these When hosts were in the prepupal stage, wasps percentages are significantly higher than the spent more time examining the host through antennal percentages of host drilling in stage L1/L2 (38.9%). drumming than in the other two stages. Host-feeding When E. tricolor was the secondary host, the host only occurred in the early larval stages (L1/L2). drilling percentage in the L3 stage was significantly higher than the percentages found for the other two stages. Subsequently, a larger percentage of hosts were found hyperparasitized in stage L3 Discussion (37.5 ± 8.3%) when compared to the early larval (L1/L2) and prepupal stages, in which only about 2% Searching and parasitization behaviour of the hosts were hyperparasitized. When the hosts of E. tricolor were E. formosa, a low percentage of hosts (14.8 ± 4.3%) in early larval (L1/L2) stages were Searching and hyperparasitism behaviours exhibited hyperparasitized, a higher percentage in the prepupal by virgin E. tricolor females were similar to beha- stage, and the highest percentage in L3. Thus, we viours observed for other, primary whitefly parasitoid conclude that L3 of both primary host species is the species such as E. formosa (van Lenteren et al. 1980) most preferred stage for hyperparasitization. and Amitus fuscipennis MacGown and Nebeker The percentages of hyperparasitism of E. formosa (Manzano et al. 2002). Our observations on E. tricolor were always higher than those of E. tricolor. show that the behavioural duration of accepting hosts Significant differences were found for all host stages (oviposition) was much longer than that of rejecting a 123 Y. Huang et al.

Fig. 5 Percentages (±s.e.) of host encountered, ovipositor drilled, male egg laid and host fed found for different host species and stages (number of host provided for each stage/ species was taken as 100%). Values between species within the same host stage are significantly different when followed by an asterisk (P \ 0.05, v2)

host after ovipositor probing. This was also observed time needed by E. tricolor to lay a female egg was for E. formosa (van Lenteren et al. 1980), and the found to be about 200–300 s (Williams 1995), which time involved in oviposition attitude can thus be used is much shorter than the time needed for laying a to determine if oviposition has occurred or not. The male egg (hyperparasitism, more than 560 s, Fig. 2). 123 Hyperparasitism behaviour

Table 1 Average percentage (±s.e.) of hosts encountered, drilled, parasitized (male egg deposition) and host-fed by virgin females of E. tricolor, when two host species were provided simultaneously (12–12) in different stages Host stage N % Hosts encountered % Hosts drilled % Hosts parasitized % Host fed upon E. formosa E. tricolor E. formosa E. tricolor E. formosa E. tricolor E. formosa E. tricolor

L1/L2 9 72.2 ± 5.6 a 75.9 ± 5.6 a 38.9 ± 3.9 a 48.1 ± 11.9 a 14.8 ± 4.3 a 1.9 ± 1.9 a 13.0 ± 3.7 a 1.9 ± 1.9 a L3 8 85.4 ± 4.9 a 77.1 ± 8.9 a 83.3 ± 5.5 b 60.4 ± 7.0 a 66.7 ± 8.3 b 37.5 ± 8.2 b 0 b 0 a Prepupa 8 85.9 ± 8.0 a 76.6 ± 6.3 a 77.1 ± 7.9 b 22.9 ± 7.0 b 43.2 ± 4.7 c 2.1 ± 2.1 a 0 b 0 a Values are significantly different when followed by different letters in the same column (P \ 0.05, ANOVA; v2 test used as post hoc test)

The same was found for other autoparasitoids, such most preferred stage for hyperparasitization for as E. pergandiella or Encarsia spp., which both spent E. tricolor is similar to information provided for more time to lay hyperparasitic eggs than primary E. tricolor by Avilla and Copland (1987). These authors ones (Buijs et al. 1981; Kajita 1989). This might be found that more males emerged from late larvae to explained by the fact that more effort has to be paid pupae than from early instar larvae (3–4 days), and by autoparasitoids to deposit a male egg inside the suggested that either a low oviposition rate or low secondary hosts, as two layers of cuticles have to be male survivorship occurred on young secondary penetrated, instead of only one as in primary hosts. A hosts. Because we have made direct observations, longer time may also be caused by a more time we can explain their results and conclude that the consuming host selection process to locate the higher number of males is largely the result of a secondary host inside the primary host. difference in acceptance of the secondary host, and Superparasitism occurred only once during all not the result of an increase in mortality. This may be observations. This indicates that virgin E. tricolor an evolutionary adaptation of autoparasitoids to females are able to discriminate between secondary decrease the risk of egg-depletion in less suitable hosts hyperparasitized by themselves, and avoid self- hosts. Old larval stages may be more suitable because superparasitism, similar to what is found for many larger hosts provide better resource for the parasi- other primary parasitoids (van Lenteren et al. 1976b; toids’ larvae to develop. For example, Liu and van Lenteren 1981; Nuffio and Papaj 2001). However, Stansly (1996) and Jones and Greenberg (1999) Hunter (1989) and Pedata and Hunter (1996) reported propose that parasitoids may immediately use host that another autoparasitoid E. pergandiella does not resources in late stages, thereby maximizing the discriminate between hosts with and without an egg. intrinsic rate of increase (r) through decreased Host-feeding only occurred during the early larval generation time, increased fecundity, or both. secondary host stages. Remarkably, the heterospecific A limited egg-load may be a factor influencing a secondary host species E. formosa was largely female’s searching intensity, oviposition rate and host preferred over the conspecific secondary host (seven acceptance (Minkenberg et al. 1992), but this could not times out of eight), but this needs to be substantiated have been of influence in our study as of all stages a as the number of observations was low. surplus of hosts was offered. In our experimental conditions an egg-limited parasitoid, as is the case for Effects of host stages on hyperparasitism E. tricolor, should maximize the quality of hosts to be parasitized and a long time is expected to select it. The Hyperparasitizing E. tricolor females encountered time spent on oviposition was always the most time both species of secondary hosts in similar numbers, consuming behavioural element during the maximum regardless the stages offered (Table 1). This indicates observation time of 2 h, as is reflected by the total time that host selection does not occur before a host has in oviposition (Fig 3; Table 2) and the percentages of been drummed, similar to those reported by van hosts drilled (Table 1). Yet E. tricolor refrained from Lenteren et al. (1976a) for the primary parasitoid egg-laying in young secondary hosts, and preferred E. formosa. Our observations that the L3 stage is the heterospecific over conspecific hosts in all stages. In 123 Y. Huang et al.

addition, the total numbers of eggs laid by E. tricolor in 2.9 our experiments was less than the maximum daily ± fecundity reported for this species (Williams 1995; Sengonc¸a et al. 2001). An experiment performed by Burger et al. (2006) with E. formosa shows similar 5.7 2.9

± results in that the time spent on oviposition occupied a large proportion of a wasps’ life time. This may indicate that wasps spent more effort on reproduction 4.4 20.6 2.7 0 0 4.1 0 0 when hosts are better for parasitism than on host- ± ± ± feeding. When oviposition and host-feeding are con- sidered as a trade-off between reproduction and survival, i.e. current and future reproduction (Jervis 4.6 19.5 3.1 29.4 3.3 9.3 and Kidd 1986; Heimpel and Collier 1996), then our ± ± ± results indicate that reproduction prevails over feeding under our experimental conditions. 0.5 21.3 0.4 47.0 0.8 39.8 Effect of host species on hyperparasitism ± ± ±

test used as post hoc test) Several other studies on selection of secondary hosts by 2 in experimental arenas with hosts of different stages and species v Encarsia autoparasitoids have been reported (Avilla 0.3 1.3 0.4 1.2 0.8 3.3 et al. 1991; Williams 1991; Bogra´n and Heinz 2002; ± ± ± Pedata and Hunter 1996). In these studies either a E. formosa E. tricolor E. formosa E. tricolor E. formosa E. tricolor E. tricolor preference for heterospecific hosts or no preference was found, but never a preference for conspecific hosts. 0.05, ANOVA;

\ The results obtained by Avilla et al. (1991) and P Williams (1991) indicated that in that case E. tricolor 1.0 1.7 1.7 1.5 0.8 3.2 ± ± ± preferred heterospecific (E. formosa on T. vaporariorum; E. inaron (Walker) on A. proletella) secondary hosts

s.e.) of virgin female over conspecific ones. No obvious preference, ± however, was found for E. pergandiella when given a choice between a combination of the conspecific host 2.7 a 3.6 1.8 ab 3.4 3.1 ac 2.3 and a heterospecific host, such as E. formosa (Buijs ± ± ± et al. 1981; Pedata and Hunter 1996) and E. hispida (as E. meritoria) (Pedata and Hunter 1996)on Trialeurodes vapoariorum. Bogra´n and Heinz (2002) showed on the other hand that E. pergandiella favoured 2.6 a 11.3 1.1 b 6.4 2.8 a 16.8 heterospecific hosts over conspecific hosts (E. formosa ± ± ± or Eretmocerus mundus Mercet) in the presence of B. tabaci as a primary host, but not when a third parasitoid species was present. What is the mechanism behind the lack or 456.7 a 20.2 431.5 a 11.1 382.5 a 25.4 presence of a certain host preference? On one hand, ± ± ± female E. tricolor may be able to discriminate between heterospecific and conspecific pupae as put

Time in arena (s) % Walking time % Standing time % Honey feeding % Antennal testing % Ovipositing time % Host feeding forward by Avilla et al. (1991), on the other hand

N other autoparasitoid species such as E. pergandiella Time budget (average percentages of behavioural elements may not (Pedata et al. 2002). Our observations showed that E. tricolor could easily discriminate Table 2 Stage L1/L2 9 6185.2 L3 8 6016.6 Prepupa 8 5822.1 Values followed by different letters in the same column are significantly different ( different host species in the prepupal stage through 123 Hyperparasitism behaviour antennal examination, but females had to insert their 1995) and E. eremicus (Hunter and Kelly 1998; ovipositor to discriminate between immature stages Briggs and Collier 2001; Hunter et al. 2002), of the hosts (Tables 1, 2). respectively. Immatures of the autoparasitoid may have specific mechanisms, such as physical defence (Gerling 1990) Implications for biological control and a different life-history strategy, to reduce and non-target effects vulnerability and protect female parasitoid larva from being self-hyperparasitized. Melanized pupae occur In this study, two host species were provided in a number of species within the genus Encarsia, simultaneously and our results suggest that the such as E. tricolor, E. inaron, E. hispida and preference to hyperparasitize by E. tricolor was E. sophia (Avilla et al. 1991; Williams 1991; Pedata directed to the hetero-specific host E. formosa.In and Hunter 1996; Hunter et al. 2002). Examining the addition, as shown by similar results of our host-ratio secondary host choice by the autoparasitoid experiment (Huang et al. 2002), selection of the E. pergandiella proved that the melanized pupal secondary host for male egg deposition greatly affects sheath in E. hispida deterred parasitism (Pedata and the sex ratio. If the autoparasitioid E. tricolor is used Hunter 1996). Immature Eretmocerus eremicus Rose in combination with E. formosa for biological control & Zolnerowich were vulnerable to parasitism by of whitefly, its behaviour to attack primary parasi- E. sophia for twice the time as were conspecific toids, may negatively influence the effectiveness of immature (Hunter et al. 2002). However, the nature control as predicted by Mills and Gutierrez (1996)in of the possible defence of a melanized pupae against their theoretical model. Data from several cage and hyperparasitism is still unclear. During our experi- field-cage experiments, designed to evaluate the ment, the presence of a melanized pupal sheath of impact of single versus multiple introductions, E. tricolor occurred soon after the prepupal stage strongly suggest the occurrence of interspecific (within one day), but in E. formosa this did not occur. competition among primary parasitoids and autopa- Therefore, two hypotheses are put forward by us to rasitoids. Recent papers give a substantial contribute explain the preference for the heterospecific host to the debate on the implications of autoparasitoids E. formosa over the conspecific host by E. tricolor: for biological control both from empirical (Hunter (1) in the process of melanization, the cuticle of the et al. 2002; Bogra´n et al. 2002) and theoretical immature, pupal E. tricolor female becomes harder (Schreiber et al. 2001; Briggs and Collier 2001) point and more difficult to penetrate and this physical of view. All these, and other papers point out that, change leads to a low attack rate (physical defence), although autoparasitoids often interact in complex (2) the immature E. tricolor female has a relative ways with primary parasitoids—often competitively higher developmental speed, and this life-history displaced, sometimes co-existent—the final outcome strategy leaves a shorter time period (vulnerability of biological control may not be necessarily disrupted window) during which it is susceptible to hyper- (Heinz and Nelson 1996; Giorgini and Viggiani parasitism compared to a heterospecific host like 2000). Most of these studies and models, however, E. formosa. Our direct observations indicate that largely include impacts within agroecosystems (pest– melanization in pupae of E. tricolor prevents them plant–parasitoids) and much less within the natural from being hyperparasitized by conspecific females. environment. The outcome may depend, however, Results from other direct observations (Loomans upon other biological characteristics, in addition to et al. unpublished) indicate that a combination of the relative suitability of the primary, whitefly host to these two factors—the presence of melanization and both autoparasitoid and primary parasitoid. the rate of this process—depend on the whitefly From the point of safety of exotic biological species involved and define the time window of pupal control agents for the indigenous entomofauna, the vulnerability to a subsequent autoparasitoid attack. A results of this study can be viewed upon from two larger window of vulnerability would largely explain viewpoints. When an exotic autoparasitoid released the apparent preference of E. tricolor and E. sophia for the control of whitefly pests in greenhouses (referred to as E. transvena) for heterospecific escapes and establishes outdoors, it might have secondary hosts as found for E. inaron (Williams serious non-target effects. When an exotic, or native, 123 Y. Huang et al. autoparasitoid that is established outdoors, subse- tricolor (Hymenoptera: Aphelinidae). Ann Appl Biol quently invades an agricultural environment like 110:381–389 Avilla J, Anado´n J, Sarasu´a MJ, Albajes R (1991) Egg allo- greenhouses, where e.g. E. formosa is effectively cation of the autoparasitoid Encarsia tricolor at different controlling the whitefly population, it might seriously relative densities of the primary host (Trialeurodes hamper biological control as shown by Del Bene and vaporariorum) and two secondary hosts (Encarsia Landi (1991) for E. tricolor and by e.g. Gabarra et al. formosa and E. tricolor). Ent Exp Appl 59:219–227 Bogra´n CE, Heinz KM (2002) Host selection by the hetero- (1999, 2003) for E. pergandiella. In such greenhouse nomous hyperparasitoid Encarsia pergandiella: multiple- conditions where many immatures of primary para- choice tests using Bemisia argentifolii as primary host. sitoid species are available that can serve as Ent Exp Appl 103:11–21 secondary hosts, and/or large number of unmated Bogra´n CE, Heinz KM, Ciomperlik M (2002) Interspecific competition among insect parasitoids: field experiments wasps are present, autoparasitoids might produce with whiteflies as hosts in cotton. Ecology 83:653–668 large numbers of male offspring (Williams 1977) and Briggs CJ, Collier TR (2001) Autoparasitism, interference, and thus disrupt biocontrol. On the other hand when an parasitoid-pest population dynamics. Theor Popul Biol exotic biological control agent, such as E. formosa, 60:33–57 Buijs MJ, Pirovano I, van Lenteren JC (1981) Encarsia per- escapes from the greenhouse into the natural envi- gandiella, a possible biological control agent for the ronment, it may encounter native autoparasitoid greenhouse whitefly, Trialeurodes vaporariorum: A study species, like E. tricolor and be strongly reduced in on intra- and interspecific host selection. Med Fac Land- numbers. The behavioural and biological traits of bouww RU Gent 46(2):465–471 Burger JMS, Huang Y, Hemerik L, van Lenteren JC, Vet LEM E. tricolor may thus reduce the risk of an exotic (2006) Flexible use of patch-leaving mechanisms in a primary species like E. formosa, or other primary parasitoid wasp. J Insect Behav 19:155–170 parasitoids naturally invading new habitats (E. hispida, Del Bene G, Landi S (1991) Biological pest control in glass- E. protransvena, E. inaron) from spreading into a house ornamental crops in Tuscany. Bull IOBC/WPRS 14(5):13–21 natural environment. There is some support for this Gabarra R, Arno´ J, Alomar O (1999) Naturally occurring hypothesis, because surveys made by Kajita (2000)in populations of Encarsia pergandiella (Hymenoptera: Japan suggest that E. formosa, when settled outside Aphelinidae) in tomato greenhouses. Bull OILB/WPRS greenhouses in which it had been released, was 22:85–88 Gabarra R, Batllori M, Albajes R (2003) Encarsia formosa and frequently attacked by native autoparasitoid species Encarsia pergandiella: addition or substraction. Bull such as E. japonica and E. sophia (as E. transvena). IOBC/WPRS 26(10):33–38 This interesting hypothesis of diminished risks in the Gerling D (1990) Whiteflies: their bionomics pest status and field when the exotic primary exotic parasitoids used management. Intercept, Andover, UK Gerling D (1966) Studies with whitefly parasites of Southern in greenhouses are attacked by native autoparasitoids California. I. Encarsia pergandiella Howard (Hymenop- certainly justifies further study. tera: Aphelinidae). Can Entomol 98:707–724 Gerling D, Alomar O, Arno´ J (2001) Biological control of Acknowledgements Y. Huang greatly appreciated a study Bemisia using predators and parasitoids. Crop Prot grant from the Royal Dutch Academy of Sciences (KNAW) to 20:779–799 accomplish her study at Wageningen University, the Giorgini M, Viggiani G (2000) A compared evaluation of Netherlands. John Thompson (Bioforce Ltd, Auckland, NZ) Encarsia formosa Gahan and Encarsia pergandiella is thanked for his information on E. pergandiella. This study Howard (Hymenoptera: Aphelinidae) as biological control was also supported by the Chinese State Key Basic Research agents of Trialeurodes vaporariorum (Westwood) and Development Plan G2000046803 and the Commission of (Homoptera: Aleyrodidae) on tomato under greenhouse in the European Communities, Agriculture and Fisheries (FAIR) south Italy. 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